Recombinant DNA methodologies capable of amplifying purified nucleic acid fragments have long been recognized. Typically, such methodologies involve the introduction of a desired nucleic acid fragment into a DNA or RNA vector, the clonal amplification of the vector, and the recovery of the amplified nucleic acid fragment. Examples of such methodologies are provided by Cohen et al. (U.S. Pat. No. 4,237,224), Maniatis, T. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, etc.
In some instances, the desired nucleic acid molecule can be readily obtained from a source material. The molecule can then be inserted into a suitable vector by either adding "linker molecules" (see Scheller et al., Science 196:177-180 (1977)) or by treating the desired molecule with a restriction endonuclease.
In other instances, however, the desired nucleic acid molecule cannot be obtained from a source material at a concentration or in an amount sufficient to permit gene cloning. In such a situation, it is necessary to amplify the nucleic acid molecule by, for example, template-directed extension, prior to introducing it into a suitable vector. Primer extension can be mediated by the "polymerase chain reaction" ("PCR"), or other means.
In the "polymerase chain reaction" or "PCR" the amplification of a specific nucleic acid sequence is achieved using two oligonucleotide primers complementary to regions of the sequence to be amplified (FIG. 1).
The polymerase chain reaction provides a method for selectively increasing the concentration of a nucleic acid molecule having a particular sequence even when that molecule has not been previously purified and is present only in a single copy in a particular sample. The method can be used to amplify either single or double stranded DNA. Reviews of the polymerase chain reaction are provided by Mullis, K. B. (Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986)); Saiki, R. K., et al. (Bio/Technology 3:1008-1012 (1985)); Mullis, K. B., et al. (Met. Enzymol. 155:335-350 (1987); Erlich H. et al., (EP 50,424; EP 84,796, EP 258,017, EP 237,362); Mullis, K. (EP 201,184); Mullis K. et al., (U.S. Pat. No. 4,683,202); Erlich, H. (U.S. Pat. No. 4,582,788); and Saiki, R. et al. (U.S. Pat. No. 4,683,194) all of which references are incorporated herein by reference).
The ability to incorporate a gene sequence into a suitable vector is typically performed using restriction endonucleases. Thus, the vector and the desired gene sequence are treated with a restriction nuclease capable of producing compatible termini which can then be ligated together to form a covalently closed vector molecule. Preferably, the restriction enzyme is selected such that its recognition site is not present in the desired gene sequence.
It would be desirable to be able to generally alter the nucleotide sequences of a desired target sequence in order to permit it to be cloned into a suitable vector without using oligonucleotide linkers/adapters, and regardless of the availability or suitability of restriction sites. The present invention provides methods suitable for accomplishing these goals.